Endothelialization of chitosan porous conduits via immobilization of a recombinant fibronectin fragment (rhFNIII7–10)

Abstract

The present study aimed to develop a pre-endothelialized chitosan (CH) porous hollowed scaffold for application in spinal cord regenerative therapies. CH conduits with different degrees of acetylation (DA; 4% and 15%) were prepared, characterized (microstructure, porosity and water uptake) and functionalized with a recombinant fragment of human fibronectin (rhFNIII ). Immobilized rhFNIII 7-10 7-10 was characterized in terms of amount ( 125 I-radiolabelling), exposure of cell-binding domains (immunofluorescence) and ability to mediate endothelial cell (EC) adhesion and cytoskeletal rearrangement. Func-K e y w o r d s :Three-dimensional scaffolds tionalized conduits revealed a linear increase in immobilized rhFNIII 7-10 with rhFNIII 7-10 concentration, Surface grafting Protein radiolabelling Protein conformation Spinal cord injury and, for the same concentration, higher amounts of rhFNIII7-10 on DA 4% compared with DA 15%. Moreover, rhFNIII7-10 concentrations as low as 5 and 20 lg ml -1 in the coupling reaction were shown to provide DA 4% and 15% scaffolds, respectively, with levels of exposed cell-binding domains exceeding those observed on the control (DA 4% scaffolds incubated in a 20 lg ml -1 human fibronectin solution). These grafting conditions proved to be effective in mediating EC adhesion/cytoskeletal organization on CH with DA 4% and 15%, without affecting the endothelial angiogenic potential. rhFNIII7-10 grafting to CH could be a strategy of particular interest in tissue engineering applications requiring the use of endothelialized porous matrices with tunable degradation rates.

Fibrin functionalization with synthetic adhesive ligands interacting with α6β1 integrin receptor enhance neurite outgrowth of embryonic stem cell-derived neural stem/progenitors

“…S3). Tubular scaffolds were prepared from squid pen chitosan (France Chitine; degree of acetylation 3.55) after purified (endotoxin levels < 0.1 EU/mL), as previously described [35]. For each experimental group, 10 female Wistar rats (10-13 weeks old) from Charles River Laboratories were used.…”

The use of new surface‐modified poly(2‐hydroxyethyl methacrylate) hydrogels in tissue engineering: Treatment of the surface with fibronectin subunits versus Ac‐CGGASIKVAVS‐OH, cysteine, and 2‐mercaptoethanol modification

“…For example, stretching of the Fn molecule can regulate the exposure of different integrin recognition sites . The cell adhesive properties of the whole Fn molecule as well as of the recombinant FnIII 7–10 fragment physically adsorbed or covalently bound to chitosan were strongly dependent on the degree of acetylation …”

“…The advantage of immobilizing the full length Fn‐monomer is the preservation of its biofunctionality, because of the presence of multiple integrin binding sites, complementary domains or domains for the binding of fibrin, collagen, heparin, syndecan, and so forth. On the other hand, it is known that Fn undergoes conformational changes upon adsorption that may lead to the loss of its bioactive properties …”

“…18 The cell adhesive properties of the whole Fn molecule as well as of the recombinant FnIII 7-10 fragment physically adsorbed or covalently bound to chitosan were strongly dependent on the degree of acetylation. 19 In the current approach, we utilize the fact that the Fn molecule exists as a dimer consisting of two nearly identical subunits cross-linked by a pair of disulfide bonds. The dimer was thus split by tris(2-carboxyethyl)phosphine hydrochloride (TCEP), resulting in two fibronectin subunits, while their ability to bind integrins and ECM components was preserved.…”

“…On the other hand, it is known that Fn undergoes conformational changes upon adsorption that may lead to the loss of its bioactive properties. 19 To characterize the bioactivity and bioadhesive properties of SIKVAV or Fn-subunits immobilized onto superporous P(HEMA-AEMA) hydrogels through the maleimide-thiol coupling reaction, the attachment, morphology, proliferation, and differentiation of MSCs were studied. In addition, the cell adhesive effects of the terminal groups of two small molecules containing sulfhydride groups, namely 2mercaptoethanol (ME) and cysteine (Cys), were also investigated.…”